Agrobacterium tumefaciens-mediated instantaneous gene transformation method for fig fruits

Abstract

The invention discloses an agrobacterium tumefaciens-mediated fig fruit transient gene transformation method, which is characterized in that a carpopodium of a fig fruit is immersed in an agrobacterium tumefaciens infection solution carrying a target gene for dip dyeing, so that the infection efficiency and the positive rate of a transgenic fruit are remarkably improved. According to the method, a carpopodium infiltration type infection system is adopted, gene delivery carried by agrobacterium is achieved through the active absorption effect of a plant vascular system, and the infection efficiency is guaranteed. Compared with a traditional fig fruit injection method, the system avoids tissue browning caused by mechanical damage, the experiment is carried out in a tissue culture room, the operation is simple, the test period is short, and the method is suitable for efficiently and rapidly verifying gene functions in fig fruits.

Description

Technical Field

[0001] The invention provides a method for transient gene transformation of fig fruit, in particular to an Agrobacterium-mediated transient gene transformation method for fig fruit. Background Art

[0002] Fig (Ficus carica L.), a member of the genus Ficus in the Moraceae family, is rich in bioactive ingredients such as anthocyanins and carotenoids, and possesses significant nutritional and economic value. The color of mature fig fruit is primarily determined by the type and content of anthocyanins and carotenoids. Influenced by these pigments, the fig peel exhibits diverse color phenotypes, including red, green, and yellow. However, as a perennial woody plant, figs have a long regeneration cycle and low transformation efficiency, making it particularly difficult to obtain stably genetically transformed fig fruit, hindering research on fig fruit quality.

[0003] Transient gene expression technology is a technique that uses specific technical means to transform genes into plants and then detect the function of the target gene in a relatively short period of time. Due to its advantages of short experimental cycles, low experimental costs, and good transformation efficiency, it has been applied in the research of various fruit trees. Currently, transient gene expression in fig fruit mainly uses the method of injecting Agrobacterium into the fruit. However, this technique is relatively complex to operate, the injection site is prone to causing browning and rot of the fruit, and it is easily affected by the external environment, resulting in low fruit transformation efficiency and unclear phenotypic observation. Therefore, a new method for validating the function of fig fruit is urgently needed. Summary of the Invention

[0004] Purpose of the invention: In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for transient gene transformation of fig fruit mediated by Agrobacterium that is non-invasive to the fig fruit and has high transformation efficiency.

[0005] Technical solution: The method for transient gene transformation of fig fruit mediated by Agrobacterium of the present invention comprises the following steps:

[0006] (1) Cultivating Agrobacterium carrying the target gene, collecting the bacterial precipitate by centrifugation, and resuspending the bacterial cells with a resuspension solution to obtain an Agrobacterium infection solution;

[0007] (2) Immerse the fruit stalk of the fig fruit in the Agrobacterium infection solution, then place the fig fruit in clean water for immersion and culture under long-day conditions.

[0008] Furthermore, in step (1), the target gene is FcMYB6, FcANS or FcCHS10, the Agrobacterium strain is GV3101, the OD value of the cultured Agrobacterium liquid concentration is 0.8, and the resuspension contains MgCl2, MES, and acetosyringone.

[0009] Furthermore, in step (2), the OD value of the Agrobacterium infection solution concentration is 0.8. The fig fruit variety is "Haile" fig, "Hardy" fig or "Brooklyn Black" fig, and the fig fruit material is unripe red fig fruit of uniform size and color that has not turned red, and the fruit stalk is retained intact. The fig stalk is immersed in the Agrobacterium infection solution, and the fruit stalk is deeply immersed in the infection solution for more than 2 / 3, and the immersion time is more than 24 hours. During the immersion process, ensure that the amount of Agrobacterium infection solution does not decrease, and add Agrobacterium infection solution in time. In addition, cover the fig fruit with a transparent plastic cover to prevent water loss. The culture temperature is 23-25°C, and after 2-3 days of long-day culture, observe and sample the fig phenotype, and evaluate the gene expression level and positive transformation efficiency.

[0010] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0011] (1) The present invention significantly improves the diffusion and infection effect of fig fruits by optimizing the Agrobacterium infection method and variety selection, significantly increases the positive rate of instantaneous transformation of fig fruits, and has high feasibility.

[0012] (2) In the present invention, the fig stalk is selected as the infection site. Compared with traditional methods, this method has the advantages that the Agrobacterium infection solution absorbed by the fig stalk is more conducive to spreading throughout the fruit, with high infection efficiency and simple operation, which significantly improves the infection efficiency of figs.

[0013] (3) This invention uses a fruit stalk infiltration infection system, leveraging the active absorption of the plant vascular system to deliver Agrobacterium-carried genes, ensuring efficient infection. Compared to traditional fig fruit injection methods, this system avoids tissue browning caused by mechanical damage. Furthermore, the experiment is conducted in a tissue culture room, which simplifies the operation and shortens the experimental cycle, making it suitable for efficient and rapid verification of gene function in fig fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The "Hale" fig fruit and the pattern diagram of fig fruit infection selected in Example 1, wherein A is the "Hale" fig fruit and B is the infection pattern diagram.

[0015] Figure 2Figures 1 and 2 show the phenotype and anthocyanin content of "Hale" figs transiently overexpressing the FcMYB6 gene in Example 1. Figure A shows the "Hale" figs after infection. The control, PRI101, is not infected with the FcMYB6 gene, and FcMYB6-PRI101 is infected with the FcMYB6 gene. The left figure shows the post-infection control treatment, the right figure shows the phenotype of the FcMYB6 gene after infection, and B shows the quantitative PCR analysis of FcMYB6 gene expression. Figure C shows anthocyanin extracts from figs after control and FcMYB6 gene infection. Figure D shows anthocyanin content in the control and FcMYB6 gene-infected figs.

[0016] Figure 3 Figure 2 shows the enhanced anthocyanin accumulation in "hardy" fig fruit by transiently overexpressing the FcANS and FcCHS10 genes in Example 2. Figure A shows the phenotype of "hardy" fig fruit after infection. The control (PFGFP) represents fruit without FcANS or FcCHS10 gene infection, the FcANS-PFGFP represents fruit infected with the FcANS gene, and the FcCHS10-PFGFP represents fruit infected with the FcCHS10 gene. Figure B shows anthocyanin extracts from fig fruit infected with the control, FcANS, and FcCHS10 genes. Figure C shows gene expression by quantitative PCR. Figure D shows anthocyanin content in the control, FcANS, and FcCHS10-infected figs.

[0017] Figure 4 The diagrams are a schematic diagram of the traditional injection method in Comparative Example 1 and a diagram of the phenotype and anthocyanin content of fig fruit transiently overexpressing the FcMYB6 gene, wherein A is a schematic diagram of the traditional injection method; B is a diagram of the phenotype of fig fruit after injection, with the left side showing FcMYB6-PRI101 injection, which is injection with Agrobacterium carrying the FcMYB6 gene, and the right side showing an empty injection, which is injection with Agrobacterium infection solution that does not carry the FcMYB6 gene; C is a diagram of anthocyanin extracts after control and FcMYB6-PRI101 infection; D is a diagram of quantitative PCR for determining FcMYB6 gene expression, and E is a diagram of anthocyanin content after control and FcMYB6 infection.

[0018] Figure 5 It is the statistical graph of positive conversion rate of the traditional injection method and the dipping method of Example 2. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below with reference to the accompanying drawings.

[0020] Example 1

[0021] The fruits of "Hale" figs that have not turned red (green fruit stage) were selected as materials; Figure 1As shown in A, as the fig fruit to be infected, the fig fruit stalk is kept intact, and the white juice flowing out of the fig stalk is wiped dry.

[0022] like Figure 1 As shown in B, the dyeing is carried out as follows:

[0023] (1) Selecting the above-mentioned "Hale" fig fruits of the same size and maturity that have not yet turned red;

[0024] (2) Select Agrobacterium strain GV3101 and transform the FcMYB6 plasmid into GV3101 competent cells through Agrobacterium transformation experiment; then cultivate Agrobacterium GV3101 bacterial solution carrying FcMYB6 gene to about OD=0.8. Collect Agrobacterium GV3101 bacterial pellet by centrifugation and resuspend it in appropriate resuspension solution (containing 10mmol / L MgCl2, 10mmol / L MES, 150mmol / L acetosyringone) to obtain Agrobacterium infection solution (FcMYB6-PRI101) with an OD value of 0.8;

[0025] (4) Place the fruit stem of the "Haier" fig fruit more than 2 / 3 deep into the Agrobacterium infection solution. Observe the infection solution every 3-6 hours to see if it decreases, and add Agrobacterium infection solution in time to ensure that the amount of Agrobacterium infection solution does not decrease. Soak for a total of 24 hours;

[0026] (5) Remove the infected "Hale" fig fruits from the Agrobacterium infection solution, place the fruit stalks in clean water, cover with a transparent plastic cover to prevent water loss, and culture in a culture room under long-day conditions (25°C, day / night, 14 / 8 h);

[0027] (6) After culturing for 2-3 days, when the “Heile” fig fruits show a distinct red phenotype, the infected figs are quantitatively analyzed and the anthocyanin content is determined.

[0028] The cells were immersed in the impregnation solution of Agrobacterium that does not carry the FcMYB6 gene (the impregnation solution was obtained under the same conditions as the original Agrobacterium GV3101) for 24 hours for comparison (empty control (PRI101)). Figure 2 shown.

[0029] Depend on Figure 2 It can be seen that compared with the empty control (PRI101), the expression level of the figs soaked in Agrobacterium-infected solution carrying the FcMYB6 gene (FcMYB6-PRI101) was significantly increased, upregulated by 6.5 times; and the overexpressed FcMYB6 gene can promote the coloring of figs, and the anthocyanin content was significantly increased, increasing by 1.5 times.

[0030] Example 2

[0031] The experimental process was the same as in Example 1, except that the fruit of "Hardy" fig was used, the vector used was PFGFP, and the target genes were FcANS and FcCHS10. The results are shown in Figure 1. Figure 3 shown.

[0032] Figure 3 Figure 2 shows the accumulation of anthocyanins promoted by transient overexpression of FcANS and FcCHS10 genes in "Hardy" fig fruit. Figure 3 As shown, when "Hardy" figs were transiently infected with overexpressed FcANS or FcCHS10 genes, the expression levels of the FcANS or FcCHS10 genes were significantly increased compared to the control. Furthermore, the overexpressed FcANS or FcCHS10 genes promoted fig coloration and significantly increased anthocyanin content. These results demonstrate that this method can be successfully used with different genes, expression vectors, and figs.

[0033] Comparative Example 1: Transient overexpression of FcMYB6 gene in fig fruit by traditional injection method to promote the accumulation of anthocyanins

[0034] like Figure 4 As shown, "Helle" figs were selected, and Agrobacterium bacteria carrying FcMYB6-PRI101 overexpression and not carrying FcMYB6 overexpression were injected into the fig peel on the tree (empty injection). The specific operation was: using "Helle" fig fruits that had not turned red as materials, gently poke a hole on both sides of the fruit symmetrically, hold the fruit with your hand, and use a 1ml syringe to inject about 50μL of Agrobacterium bacteria carrying FcMYB6 overexpression and not carrying FcMYB6 overexpression into one side of the fruit. When the "Helle" fig fruit showed a clear red phenotype after 7-10 days of culture, the infected figs were quantitatively analyzed and the anthocyanin content was determined. The results are as follows Figure 4-5 shown.

[0035] Depend on Figure 4 As shown in Figure B, injection of FcMYB6 gene-overexpressing Agrobacterium also caused figs to turn red, indicating successful acquisition of positive figs. However, some of the injected figs showed browning and rotting at the injection site, indicating that this technique is susceptible to environmental conditions.

[0036] Depend on Figure 5 It can be seen that the positive success rate of the infection method of Example 1 is 56%, while the positive success rate of the traditional injection method of Comparative Example 1 is 20%.

[0037] It can be seen that the novel infection method for Agrobacterium-mediated transient transformation of fig fruit proposed in the present invention significantly improves the color change effect of the injected fruit and the positive rate of transient transgenic figs by optimizing the infection method of figs and the selection of multiple varieties.

Claims

1. A method for transient gene transformation of fig fruit mediated by Agrobacterium, characterized in that: The following steps are involved: (1) Cultivating Agrobacterium carrying the target gene, collecting the bacterial precipitate by centrifugation, and resuspending the bacterial cells with a resuspension solution to obtain an Agrobacterium infection solution; (2) Immerse the fruit stalk of the fig fruit in the Agrobacterium infection solution, then place the fig fruit in clean water for immersion and culture under long-day conditions.

2. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (1), the target gene is FcMYB6, FcANS or FcCHS10, the Agrobacterium strain is GV3101, and the OD value of the cultured Agrobacterium liquid is 0.

8.

3. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (1), the resuspension contains MgCl2, MES, and acetosyringone.

4. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (2), the OD value of the Agrobacterium infection solution concentration is 0.

8.

5. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (2), the fig fruit is a variety of "Hale" fig, "Hardy" fig or "Brooklyn Black" fig.

6. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (2), the fruit material is unripe red fig fruits of uniform size and not yet reddened, with intact fruit stalks.

7. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (2), the base of the fruit stalk is immersed in the Agrobacterium infection solution, and the fruit stalk is immersed in more than 2 / 3 of the Agrobacterium infection solution, and the immersion time is more than 24 hours.

8. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (2), during the infection process, ensure that the amount of Agrobacterium infection solution does not decrease and add Agrobacterium infection solution in a timely manner.

9. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (2), after dipping, the fig fruits are covered with a transparent plastic cover to prevent water loss.

10. The method for transient gene transformation of fig fruit mediated by Agrobacterium according to claim 1, characterized in that: In step (2), the culture temperature is 23-25° C., and phenotypic observation is performed after 2-3 days of long-day culture, and the gene expression level and positive transformation efficiency are evaluated.

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